Construction Of Prokaryotic Expression Vector PTWIN1-EgAgB8/3and Production Of Anti EgAgB8/3Polyclonal Antibodies And Evaluate Its Diagnostic Value

Objectiv: To clone a gene fragment encoding EgAgB8/3antigen, Construction ofprotein self-splicing prokaryotic expression vector pTWIN1-EgAgB8/3, and express toobtain pure recombinant antigen of Echinococcus granulosus AgB8/3(rEgAgB8/3), thento prepare anti rEgAgB8/3polyclonal antibodies for establishment of EgAgB8/3nativetargeted sandwich-ELISA coproantigen test for dogs infected with Echinococcus spp, andevaluation of its diagnostic value. Methods: The specific primers were designedaccording to published nucleotide sequence of EgAgB8/3, deposited in the Genbankdatabase(AF362442). The nucleotide sequence corresponding to the secreted form ofEgAgB8/3was amplified by PCR, pEASY-T1-EgAgB8/3recombinant plasmid wasconstructed and confirmed by enzyme digestion and sequencing. The target gene wasdirectionally cloned into pTWIN1(+) for prokaryotic express. The recombinant plasmidpTWIN1-EgAgB8/3was transformed into E.coli DH5α, and the positive clones screenedby Ampicillin，identified by restriction endonuclease analysis and PCR amplification；andtransformed into E.coli ER2566recombinan protein expression. The fusion proteinCBD-intein1-EgAgB8/3was expressed by inducing with IPTG.. The target proteinrEgAgB8/3was purified by chitin binding affinity purification column. The expressionproduct and the target protein were analyzed by SDS-PAGE and western blotts. Animalswere immunized with purified rEgAgB8/3, Total serum IgGs from immunized animalswere purified by ammonium sulfate precipitation method. A batch of purified IgGs werelabeled using sodium iodide method, In Sandwich-ELISA system, the unlabeled IgGs were used as coating antibodies the crude protein extract from dog feces infected withEchinococcus spp were used as sandwich antigens, and the labeled IgGs were used as finalconjugate. Results: The target gene fragment was successfully cloned and ligated intopTWIN1to form proteim self-splicing prokaryotic reconbinant expression vectorpTWIN1-EgAgB8/3. SDS-PAGE and western blott analysis showed that the fusionprotein CBD-intein1-EgAgB8/3was expressed as soluble protein. The chitin bindingdomain (CBD) and intein1were removed from target protein rEgAgB8/3at one stepduring the chitin column affinity purification. High purity EgAgB8/3recombinant proteinand the high titer mono-specific polyclonal antibodies were successfully prepared. Thesandwich-ELISA coproantigen detection system Revealed93.1%sensitivity and97.6%specificity to detect EgAgB8/3native antigen in feces of dogs infected with Echinococcusspp. Conclusion: The high level of soluble target protein rEgAgB8/3with few additionalaminoacid residues was successfully purified by simple treatment, after expression, thismay allow us to keep the original amino acid sequence and pssible activity of the targetprotein, and the polyclonal antibodies obtained by animal immunization revealed highsensitivity and specificity in sandwich-ELISA coproantigen detection system. This allowsus to overcome the nonspecific cross-reactivity of sandwich-ELISA coproantigendetection system with other dog intestinal helminthes due to the coexpressed fusionpartner. However, the stability and promotional use this method needs further evaluationusing epidemiological mass data.